A simple protocol for the isolation of amentoflavone from two species of Biophytum DC. (Oxalidaceae) and evaluation of its antiproliferative potential

Abstract

The biflavonoid, amentoflavone, isolated earlier from the medicinally important species, Biophytum sensitivum, is reportedly a potent bio-active compound. In the present study, extraction protocols were standardized for the isolation of amentoflavone from two other species of Biophytum viz. B. reinwardtii (Zucc.) Klotzsch and B. veldkampii Shanavas et al and subsequent characterization of the same (using various chromatographic and spectroscopic methods) followed by screening of its bioactive potential using selected in vitro cytotoxicity assays (MTT assay, Trypan Blue Exclusion Assay and Flow cytometry).The shade-dried plant parts of the two species of Biophytum were Soxhlet extracted with ethyl alcohol (70%) and used as the raw material for the isolation of amentoflavone. Column chromatography, with a gradient solvent system of chloroform, methanol and water, followed by the thin layer chromatography of the pooled fractions with a solvent combination of chloroform, acetone and formic acid was adopted. A single fraction in both species, showed a clear prominent band in TLC, [a distinct fluorescent yellow band under short UV (100 nm) and blackish brown colour under long UV (320 nm)]. The band crystallized into a yellow powdery substance. Co-TLC (Rf value- 0.6) and High Performance Liquid Chromatography (Rt- 5.3 min) was used to confirm the isolated compound as amentoflavone. Yield, physical (solubility and melting point) and chemical properties Ultraviolet- Visible spectroscopy (UV–vis), High Resolution Mass Spectroscopy (HRMS) and Nuclear Magnetic Resonance Spectroscopy (1H, 13C, DEPT) of amentoflavone were also noted for both species. The yield of amentoflavone was higher in B. reinwardtii (86.66 mg /100 g dried plant powder) than in B. veldkampii (77.50 mg/100 g). The data generated from the different spectral techniques corresponded well with the chemical configuration of amentoflavone in the database. Therefore it could be confirmed that the isolated pure compound from both species of Biophytum was amentoflavone (C30H18O10). The isolated compound, amentoflavone was tested against cancer and normal cell lines. Amentoflavone was not cytotoxic to Human Peripheral Lymphocytes (normal cell lines) in trypan blue exclusion assay. MTT assay clearly showed that the exposure of amentoflavone at different concentrations resulted in decrease of MCF- 7(cancer cell lines) cell proliferation in a dose- dependent manner. Apoptosis was found to be dose-dependent, which was further confirmed by flow cytometry. Significant cell cycle arrest occurred in S phase and G2/M phase. Thus, anticancer studies (trypan blue exclusion assay, MTT assay and flow cytometry) proved useful for the scientific validation of the anticancer activity of the isolated pure compound, amentoflavone. The present study is the first report on the isolation of amentoflavone from Biophytum reinwardtii and B. veldkampii. Since the amentoflavone content in the two species of Biophytum is high, it can be extracted in sufficient amounts and both species can be considered potential source of the compound.